Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning ¹H NMR spectroscopy

Abstract

High-resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi-core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9- and 1.3-ppm signals) and metabolite quantification using HR MAS H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16-0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one-way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.

Imaging breast cancer using hyperpolarized carbon-13 MRI

“…Previous studies showing that intertumoral metabolic heterogeneity is more pronounced than intratumoral metabolic heterogeneity (32) and that core biopsy samples can be used to reliably assess intertumoral differences (33)(34)(35)(36)(37) provided us with a rationale for comparing global imaging-based metrics (mean LAC/PYR and mean summed SNR LAC ) with the results from IHC and RNA sequencing of single tumor biopsies. The strong correlation between the LAC/PYR with tumor volume, which is known to correlate with hypoxia (38), led us to investigate the contribution of hypoxia to the measured lactate signal.…”

Hormone-Independent Mouse Mammary Adenocarcinomas with Different Metastatic Potential Exhibit Different Metabolic Signatures

“…Metabolic tumour compartmentalisation has been investigated, either in vivo and/or ex vivo, in lymphoma (different lipid markers found to characterise necrotic and non-necrotic tumour regions [ 22 ]), liver cancer (different metabolic profiles associated with distinct intra-tumour immune statuses) [ 26 ], lung cancer (glucose levels differing between higher and less perfused tumour areas) [ 27 ], sarcoma (specific lipid/protein signatures correlated with different grading characteristics within the tumour) [ 24 ], kidney cancer (different metabolic patterns associated with tumour portions with distinct drug sensitivity) [ 28 ] and BC (differential phospholipid intra-tumoural distribution possibly related to different degrees of proliferation, hypoxia and inflammation) [ 23 ]. Besides the latter study, based on mass spectrometry (MS) imaging [ 23 ], intra-tumour metabolic heterogeneity in BC has been assessed by direct analysis of the tissue using high resolution magic angled spinning (HRMAS) nuclear magnetic resonance (NMR) [ 29 , 30 ]. Central and peripheral tumour samples showed variability in phosphocholine (PC) and phosphoethanolamine (PE) contents, whereas central specimens and core biopsies showed variability in adipate, arginine, fumarate, glutamate, PC and PE.…”

Highlight report: Intratumoral metabolomic heterogeneity of breast cancer

“…Recently, Mikheil Gogiashvili and colleagues from TU-Dortmund have published a study about the metabolomics heterogeneity of breast cancer (Gogiashvili et al, 2017[ 8 ]). The background of this study is the practically relevant question, whether measurement of a single biopsy is sufficient when analyzing tumors from a cohort of patients.…”

“…Therefore, the authors performed multi-core sampling of six small specimens from individual tumors and quantified 32 metabolites. Not unexpectedly, the intertumoral differences were larger compared to intratumoral differences (Gogiashvili et al, 2017[ 8 ]). More importantly, a random forest- classifier trained on a sample set of individual tumors correctly predicted tumor identity of an additional set of independent cores from the same tumors (Gogiashvili et al, 2017[ 8 ]).…”

“…Not unexpectedly, the intertumoral differences were larger compared to intratumoral differences (Gogiashvili et al, 2017[ 8 ]). More importantly, a random forest- classifier trained on a sample set of individual tumors correctly predicted tumor identity of an additional set of independent cores from the same tumors (Gogiashvili et al, 2017[ 8 ]). Therefore, the study shows that despite the intratumoral heterogeneity the analysis of only one or few replicates per tumor can be justified.…”